Wood Preservation Based on Neem Oil: Evaluation of Fungicidal and Termiticidal Effectiveness Gilmara de Oliveira Machado Wagner L. Polito Francisco Antonio Rocco Lahr André Luis Christoforo Carlito Calil Junior Laurie J. Cookson Marcio Rogério da Silva Abstract This research examined oil from the neem tree (Azadiracita indica A. Juss.) for its potential as an eco-friendly wood preservative. In contrast to expectations from the literature, and fun in the bioassays. Using standard experiment 1.0, 2 concentrations of 0.01, 0.1 and 1.0 percent ne neem oil can only be useful as a wood preserv with cobiocides. -eording to which neem oil should be effective against neem oil performed poorly as a preservative for Pinus radiata D. Don wood, W procedures from the wood preservat and 5.0 percent neem oil in white spirit were bioassayed against five speci n oil were hio assayed cif new, optimized formulations are soug! sects h suffered significant mass inst two species of termites. It is concluded that probably exploiting synergy Woo ic natural materia containing close and hemicelluloses, and can therefore act as food for a group of extremely active organisms, the xylophages, whose activity leads to both the decomposition of substratum and the reduction of material to its constituent elements. The xylophages of greatest economic importance are fungi and termites. Owing to the problems associated with wood biodeterioration, especially in tropical countries, treatment techniques using preservatives are often required if wood is to be used in construction (Cavalcante 1982, Costa 2000), Goncems have been raised about the traditional pre vatives used to protect wood because of the risk they may pose to both the environ particular concern are preservatives based on heavy metals such as chromium and arsenic, and those containing fluorine or ereosote. Because the toxie compounds used often have low biodegradability, additional difficulties can arise in the post-use disposal of treated timber (Evans 2001, Hata et al. 2006) ‘A more environmentally friendly alternative may occur through the use of natural biocides, and some researchers have focused on the chemical components from the extracts of wood and other plants (Ohmura et al. 2000, Cookson et al, 2004, Rodrigues et al. 2012, Syofuna et al. 2012). This study considers the use of oil from the (Azadirachta indica A. Juss) as a potential eco-friendly ‘wood preservative. Neem oil has low mammalian toxicity, while possessing biologically active compounds, such as n tree 202 azadirachtin and other limonoids (Kraus 1995), Azadirach- tin is a highly oxidized tetranontriterpenoid that has antifeedant and growth regulating effects on insects and fungi, although its biochemical effects at the cellular level are still not understood (Mordue and Blackwell 1993, Islam cet al. 2009). Neem oil and extracts have been reported to be active st termites, including Zoorermopsis nevadensis. (Ha- ‘gen) (Ohmura et al. 2006), Reticulitermes speratus Kolbe (Serit et al. 1992), Coptotermes formosanus Shiraki (Grace and Yates 1992, Doolittle et al. 2007), and Incisizermes “The authors are, respectively, Research Scientist, School of Forest Engineering, Univ. of Centro-Oest, Riozinho district, Irati, Parana, Brazil (gilmaramachado“@yahoo.com.bry; Research Scientist, School ‘of Biological Sci, Monash Univ., Clayton, Victoria, Australia yaensisi.com); Research Scientist, School of Me- Engincering, Univ. of Sio Joo del-Rei (UFSI), Sio Joo -Rei, Minas Gerais, Brazil (alchristofaro@yahoo.com br (corre- sponding author)); and Research Scientist, Chemical Inst. of Sio Carlos. (polito@igse.usp.br), and. Research Scientist, Research Sciemist, and Research Scientist, Wood Structural and Wood Lab. (Lamem), Engineering School of Sio Carlos (marciome@se.usp br, calil@sc.usp.b, froeco@ s.usp.br), Univ. of Sio Paulo, S30 Carlos, Sio Paulo, Brazil. This paper was received for publication in May 2013. Article no, 13-00050, (Forest Products Society 2013 Forest Prod. J. 63 5/6):202-206, oi:10.13073/FPJ-D-13-00050 MACHADO ET AL.‘marginipennis (Latreille) (Arcos-Roa et al. 2001), Methanol and hexane extracts from a similar timber species, izadirachta excelsa (Jack) Jacobs, were also active against Coptotermes curvignathus Hohngren (Sajap ct al. 2006). Neem oil impregnated into Populus deltoides Bartr, ex Marsh controlled Microcerotermes beesoni Snyder in a Iaboratory bioassay (Dhyani and Tripathi 2006) and was Suggested as a spray treatment that could avert termite attack (Yashroy and Gupta 2005). Neem wood or mulch itself has some resistance t© C. formosanus (Delate and Grace 1995) and in India is usually free from insect attack (Koyani et al. 2011). ‘Neem also proxiuces fungicidal compounds (Locke 1995, Paul and Sharma 2002, Amadioha 2004). ‘The outer heartwood of neem was found to be naturally durable against four species of basidiomycete decay fungi (Rao 1990). High concentrations (25%) of neem oil impregnated into Pinus roxburghit Sargent controlled the white-rotting fungus Trametes versicolor (L.: Fr.) Pikit and the brown- rotting flngus Postia placemia (Fr.) M. Larsen & Lombard (Dhyani and Tripathi 2006). Neem oil i usually produced by crushing seeds and is an available commercial product in Brazil. To examine whether commercially available neem oil could be used as ‘a wood preservative, we dissolved it in white spirit, which is a common solvent for the timber treatment industry, This formulation was assessed against termites and decay fungi using laboratory bioassays that are standard test procedures for new wood preservatives in Australia (Australasian Wood Preservation Committee [AWPC] 2007). Materials and Methods Termite bioassays Solutions of 0.01, 0.1, and 1.0 percent (m/m) neem oil in white spirit were impregnated into sapwood specimens of Pinus radiata D. Don, 50 by 25 by 15 mm. The acem oil was obiained from a farm plantation in Si Paulo state, Brazil, where it was collected from crushed seeds. Treatment was achieved by pulling a vacuum of ~98 kPa for 30 minutes, adding the treatment solution under vacuum, releasing the vacuum, and then leaving the specimens to soak at atmospheric pressure for 30 minutes. The specimens were removed, blotted to remove excess solution, weighed to determine solution uptake, and wrapped in plastic bags, ‘where they remained for | week to slow the rate of solvent evaporation. Specimens were then placed on trays in the laboratory to air-dry for 4 weeks. They were artificially weathered in vacuum ovens for 5 days at ~98 kPa and 40°C removed to cool in a desiecator, and weighed to determine the ovendry mass. Additionally, the same procedures were used with fest specimens that were submitted to two solvent control treatments (water alone and white spirit alone). Test specimens were also treated with the traditional preservative copper-chromium-arsenic (CCA) to the approved hazard class 2 retention of 0.32 percent (nvm) total active elements (TAE) of Cu, Cr, and As (AS 1604.1, Standards Australia 2005). There’ were six replicates per termite species and retention. The test termites were Copiotermes acinaciformis (Froggatt) and Masiotermes: darwiniensis Froggatt, which were obtained from the Northern Territory near Darwin, Australia, For the C. acinaciformis bioassay, a single test specimen was embedded in a moist matrix of Coptotermes FOREST PRODUCTS JOURNAL Vol. 63, No. Si6é acteus (Froggatt) mound material (SO g, 80% moisture content [MC within a |.2-liter glass jar. Ten grams of C dacinaciformis was added to each jar. A Bakelite lid, with a central 9-mm-diameter ventilator, closed the jar. The duration of the bioassay was 8 weeks. The M. darwiniensis bioassay utilized 700-mL glass jars in which a mixture of 18 g of vermiculite (5- to 10-mm particle size range) and 6 g of Eucalyptus regnans F. Mucll. sawdust was placed. A single test specimen was. then embedded upright in the vermiculite-sawdust matrix close to the inside wall of each jar. Sixty-six milliliters of water was then added to the mairix to achieve 275 pereent MC. Fifteen grams of M. darwiniensis was added to each jar, and ‘metal lid, with a central 9-mm-diameter ventilator, closed the jar. The duration of the bioassay was 6 weeks. ‘At the conclusion of the bioassays, test specimens were removed from the jars and cleaned. Test specimens, as well as vacuum oven controls, were then vacuum oven-dried tunder the same conditions used to obtain the initial mass Affer cooling and weighing the test specimens, the final and initial masses were compared to determine the percentage of mass loss. Fungal bioassays Solutions of 0.01, 0.1, 1.0, 2.5 and 5.0 percent (n/m) neem oil in white spirit were’ impregnated into P. radiata sapwood specimens 20 by 20 by 10 mm using the same procedures as described previously. The 5.0 percent concentration was close to the maximum amount of pure neem oil that could be dissolved in white spirit. For the CCA. comparison, test_ specimens were treated to the approved hazard level 3 retention of 0.38 percent (mim) TAE (AS 1604.1, Standards Australia 2005). Vacuum oven- drying was used to artificially weather the test specimens as described in the termite bioassay. In addition, another set of test specimens treated with 1.0, 2.5 and 5.0 percent (m/m) neem oil was leached before vacuum oven-drying. These were vacuum impregnated with water and leached with more than three times their volume of water in jars on a shaking water bath at 35°C for 5 days, with the water changed daily. After obtaining initial masses following the vacuum ovendrying. procedure, all test specimens were sterilized by gamma irradiation. ‘The test vessels for the fungal bioassay were 250-mL. ap glass jars each containing 150 z of “Toolangi loam” soil moistened to 60 percent MC. Two poplar ipwood veneer feeder strips, previously soaked overnight in | percent malt extract solution, were placed on the soil ‘each jar. Jars were autoclaved for 2 hours. The feeder strips were inoculated with the appropriate test fungus, i.e brown-rotting fungi, Coniophora olivacea (Fr.) Karst Fomiiopsis lilacino-uilva (Berkeley) J. Wright and De- schamps, Serpua lacrymans (Schum. ex Fr.) SF. Gray, and Glocophyltum abjetinum (F.) Karst, and the white-roiting fungus Perenniporia tephropora (Mont.) Ryv. One set of jars was left uninoculated as a sterile control to determine ‘whether there was any mass loss or gain not attributable to fungal attack, Alter 14 days, the fungi had grown sufficiently on the feeder strips (30 days for S. lacrymans), whereupon sterilized test specimens were planted. There were six replicates. per fungus-retention combination. Fungi and sterile controls were incubated at 25°C, with the exception of S. Jacrymans, which was incubated at 20°C. Relative 203humidity was at least 85 percent, because jars were placed in trays containing water, and each tray was enclosed in a large plastic bag. After 12 weeks of incubation, specimens ‘were removed from the jars, weighed to determine MC, vacuum oven-dried, and weighed once more to obtain individual mass losses. The toxic threshold value is considered to have been achieved when the mean mass loss is less than 3 percent for fungi, or less than 5 percent for termites. Statistical analyses Analysis of variance (ANOVA), with significance level of| 5 percent, and taking the equivalence between means as null hypothesis (Hp: = Ha =. . .= Hy), evaluated with the aid of SAS software, was used to verify the effect of treatments fon mass loss of the woods. P Values greater than the significance level implies accepting Ho, rejecting it ‘otherwise. To validate the ANOVA model, the normality data and equivalence between variances were verified, using the Anderson-Darling and Levine's tests, respectively, both at the 5 percent significance level, with consideration of normality data and equivalence between variances as the null hypothesis for the tests, When treatments proved to have a significant effect on mass loss according to ANOVA, Tukey's test was used for grouping. Results ‘The mean mass losses caused by C. acinaciformis and M. darwiniensis to test specimens are shown in Table 1. Both termite species caused extensive damage to the water and white spirit controls, with mean mass losses above 90 pereent for C. acinaciformis and above 99 percent for WM. darwiniensis. In comparison, the specimens treated with hazard class 2 retention of CCA had negligible mean mass Joss, demonstrating excellent termite control. Almost al test specimens treated with the 0.01 and 0.1 percent solutions had mean mass losses similar to the untreated controls, However, those treated with the 1.0 percent neem solution had slightly reduced attacks compared with the controls (P 0.05), with 59.9 percent mean mass loss against C ‘acinaciformis and 72.5 percent mean mass loss against M. darwiniensis, demonstrating. slight termite resistance. In- ‘deed, all termites exposed to the 1.0 percent neem treatment Table 1—Mean mass loss in Pinus radiata test samples Subjected to bioassay against Coptotermes acinacitormis. and Mastotermes darwiniensis. “Mean * SD mass loss Treatment .acinacifrmis—-M. darwiniensis Water B62 82A 9.72 00A White spirit alone 292694 9.72000 c 03202D 342 14€ White spirit with neem, % (nm) ‘Neem, 0.01 86621418 972000 Neem, 0. SLO=68A 99.7 = 008 Neem, 1.0 B9Z110C 2S= 6B * CCA = copper-chromium-arsnic. The mass losses are adjusted for vacuum oven corto, Within the same column, means followed by the same leter are not sigriianty diferent (Tukey test at $%) 204 had lied by the end of the bioassay, while the majority of termites in jars containing test specimens treated with the 0.01 or 0.1 percent neem solutions were alive. This result suggests that a higher concentration of neem could give a higher level of protection against termites. Table 2 shows the data for mean mass losses caused by the decay fungi C. olivacea, F. lilacino-gilva, S, lacrymans, G. abietinum, and P. tephropora. The treatments with water and white spirit had no clear inhibitory effect upon the ‘growth of all brown-rotting fungi, because mean mass losses were high, in the range 35 to 65 percent. White-rotting fungi usually cause lower mass losses in sofiwoods than brown- rotting fungi in bioassays, and this occurred with P. tephropora, where the mean mass loss was 19. percent (Table 2). These results with the untreated controls demonstrate the viability of the bioassay methods used. ‘The treatment with CCA gave the best protection against all test fungi, because mean mass losses were below the toxic threshold of 3 percent (Table 2). In contrast, test specimens treated with 0.01, 0.1, 1.0, 25 oF S.0 percent (mw! 1m) neem in white spirit were heavily decayed and generally incurred similar mean mass losses to the untreated controls at all concentrations examined. Even the highest concen- tration of neem (5.0%) failed to protect P. radiata (Table 2). ‘There was a small trend in unleached blocks for lower mean :mass losses with higher treatment concentrations against C. olivacea and S. lacrymans (Table 2). Nevertheless, the treatments investigated had a negligible or minor ability to provide protection against fungal attack. P. radiata samples treated with 1.0, 2.5 and 5.0 percent neem concentrations \were also examined after artificial leaching, The mean mass losses in the majority of leached specimens were not significantly different from, or significantly higher than (P > 0.05), the corresponding test specimens without leaching, although there were some minor exceptions against G, abietinum and P. tephropora (Table 2). Note that the distribution shows normality, and equivalence of variances was observed (P > 0.05), validating the results from ANOVA. Discussion In contrast to previous reports in the literature of neem oil being a promising source of biocides, the results from this research suggest that neem oil is ineffective as a wood preservative against termites and decay fungi at the treatment levels examined, Our studies were conducted by impregnating softwood specimens and followed the stan- dard procedures used for evaluating the efficacy of new ‘wood preservatives in Australia (AWPC 2007). These are quite rigorous tests, where test specimens are vacuum oven dried (H2 tests) or leached and vacuum oven-dried (H3 tests) t0 simulate long-term ageing in service, a process that ‘may have removed active neem components. These results corroborate the findings by Grace and Yates (1992), who found lower activity against termites (C. formosanus) compared with other insect species in filter paper trials of commercial neem-based insecticide. Additionally, Paes et al, (2012) showed that formulations based on alcoholic solutions of neem failed to protect impregnated wooden stakes when exposed to decay fungi in soil contact. Despite the poor results for neem oil, there may still be ‘merit in optimizing formulations to use it as an eco-friendly wood preservative. It is worth noting that our results showed that with neem oil at 1.0 percent, attack by termites was MACHADO ET AL,Table 2—Eticacy of treatments against wood decay fungi. ‘Mean = SD mass loss (6) Contophora olvacea_ Fomitopss Macin-gibva ‘Treatment Water 684 = 104 380 = S8E White spint alone 64 = 22 3562 61E cca 1313D 03 =03F ‘White spirit with neem, % (nvm) Neem, 0.01 650254, M17 2896 Neem, 0.1 12528 aba = 290 Neem, 10 00 +388 4542388 Neem, 25 5972458 4532398 Neem, 50 557 = 40 403 = 43D Neem L, 10 6122498 SSI =60A Neem L235) 628 +286 SLL=32A Neem, 50 61s = 508 4552388 * CCA copperchromiun-arsenic: L = lashed _ "Thema not signiicamly different (Tukey test at $%) reduced, and the termites had died by the end of the bioassay, This result suggests that a higher concentration of neem oil could give a better level of protection against termites, possibly leading to suitable levels of preservation Other formulations of neem may be more effective, or neem may offer synergies with cobiocides (Antwi-Boasiako and Damoah 2010). For instance, Venmalar and Nagaveni (2005) formulated neem with copper and obtained control against white- and brown-rotting fungi. Certainly, further studies are warranted to obtain a better understanding of neem oil’s biocidal potential Acknowledgments The authors would like to acknowledge Fundagio de Amparo a Pesquisa do Estado de Sio Paulo (FAPESP, a Brazilian government funding agency) for the financial support given to this research and the CSIRO for providing, laboratory support. We would also Tike to thank Jenny Carr, Narelle Chew, and Damian Scown for their helpful assistance with the bioassays. Literature Cited Amadioha, A. C. 2004, Contol of blank rot of potato caused by Rhizoctonia btaiculawsing some plants exacts, Arch, Phytopathol. Plant Prot. 37-11-11 ‘Antwi-Boasiao, C. and A. Damoah, 2010, Investigation of synergistic effects of extracts from Exohrophleum suaveolens, Azadirachta indica, and Chromolacna odorata on the durability of nnaris toxcari. nt. Binder. Biodegrad. 6497-103, Arwos-Roa, J, J. T. Mendez-Monticl, and R. Campos-Bolanos. 2001 feoto del acite de Nim azadirahta indica juss sobre la termita de ‘madera seca Incisitermes marginipennis(Lateile) Usoptera: Kalo- termtidae), Rev. Chapingo 7:139- 143. Australasian Wood Preservation Committee (AWC). 2007. Prowacols for assessment of wood preservatives, Austaasian Wood Preservation Commitee. AWPC-Ensis, Clayton. itp www.ia.coma. Ace cessed August 20, 2013, Cavalcante, M. S. 1982, [Biological deterioration and wood preserva- tion), Technological Research Insitute, Sao Paulo. 40 pp. Cookson, L. J, R. de Nys, GP. Steinberg, and N. Chew. 2008, Wood Preservation using furanones derived from marine algae. IRGWP/ 14-10806, ‘The Intemational Research Group on Wood Protection, Stockholm, Costa, A. M. L. 2000, Cupins-praga: Morfologia, Biologia © Controle. Edifub, Rio Claro, FOREST PRODUCTS JOURNAL VoL. 63, No. 5I6 Serpulalarymans Gloom abletinum _Perenniporta ephvopora 382 =82A 242358 WS =47A 43=28A, S692 578 193 =S8A 09 = 05D 01 02D 01 £026 3982440 519 = 12.0 BC Is = 338 272 = 488 272908 166 = 41 274538 5392668 2+ 43a 264 = 358 SOE 47A 205 £264 198 * 66C 607 S74 203 = 368 309 = 42.8 4802970 252240 2722838 532247 166 237¢ 283 243 B 5222688 2241 AB ess ar the results after 12 weeks of incubation and are adjusted for sterile controls. Within each column, means followed by the sme letter are Delate, K. M. and J. K, Grace. 1995, Potential use of pathogenic fungi its to control the Formosan subteranean termite, Coprotermes Jormosanus Shitahi (Isoptera: Rhinotermitidas), J. Appl. Entomol 119:93-95 Dhyani, 8. and S. Tripathi, 2006. Protection of hand and softwood through Neem leaves extracts and oil: A direction towards develop- ment of evorftiendly wood preservatives. IRGIWPI06-303%4. The Intemational Research Group on Wood Protection, Stockholm. Doolitle, M.A. Raina, A. Laxand, and R. Boopathy. 2007, Effect of tural products on gut cndosymbiotic microbes in formosan subterrancan termites. I, Biodeter, Bodeprd. 39:69-71, Evans, F. G. 2001. Restriction for use and waste management for ‘pressure treated wood, The curtent situation in Norway. IRGIWP? (01-5017S, The Intemational Research Group on Wood Protection, Stockholm, Grace, J. K. and J. R, Yates, 1992, Behavioral effets of noc insecticide on Coptorermes formosanus (Isoptra: Rhinotermitdae), Trop. Pest Manag. 38:176- 180. Hata, TL. J Cookson, ¥-C. Jang, B. Tarakanadha, ¥. Imamura, $.N. Kartal, and T: Shibata, 2006, The production and management of ‘wood treated with chromated-eopper-arserate in Asia and Oceania with emphasis on Australia, India, Japan, and Korea. dn: Environ ‘mental Impacts of Treated Wood. T. Townsend and H. Solo-Gabriele (Eds). CRC Press, Boca Raton, Fora. pp. 59-73, Islam, M., I. Shams, G. N. M. Ilias, and O. Hannan, 2009, Protective antifungal effect of neem (Azadirachta indica) extracts on mango (Mfangijera indica) and rain wee (Albizia saman) wood, tn. Blodeter odegrad. 63241243, Koyani, R-D. G. V. Sanghvi, and K.S. Rajput 2011. Comparative study ‘on the delignification of Azadirachta indica (L) Del, wood by CCirysoxporium asperarum and Trichoderma hasianum. dn. Biodeter Biodegrad. 65:179-184 Kraus, W. 1995, Biologically active ingredients. In: The Neem Tree Sources of Unique Natural Products for Integrated Pest Management, Medicine. Industry and Other Purposes. H. Schmutterer (Ed). Weinhsim, New York. pp. 35-88 Locke, J.C. 1995. Fung. In The Neem Tree: Sources of Unique Natural Products for Inicgrated Pest Management, Medicine, Industry and ‘Other Purposes. H. Schmutterer (Ed). Weinieim, New York. pp. 118 i Monduc, A. J. and T. A. Blackwell, 1993, Azadirachin: An update, J Insect Physiol. 39:903-924, Ohmura, W., 8. Doi, M. Aoyama, and S. Ohara. 2000. Feeding preference substances in steamed Japanese larch (Lari lepilepis) 4. Wood Set, 6:199-183 ‘Ohmura, W., M. Oztki, and R Yamacks, 2006, Behavioral and electrophysiological investigation on test response of the termite 208Zootermopsis nevadenss to wood extractives, J. Wood Sci. $2:261 264 Paes, J. B., A. D. de Souza, C. R. de Lima, and P.F, de Souza. 2012. Efficiency of neem (Azadirachta indica A. Juss.) and castor off plant (Ricinus communis L.) oils for the improvement of Ceiba pentranda (L) Gaenh wood resistance to xylophagous fungi in soil bed test ine. Florest. Santa Maria 22:611-624, Paul, P. K. and P. D. Sharma, 2002, dzadirachta indica leaf extract induces resistance in barley agaist leaf spe disease, Physiol, Mol Plant Pathol 61:3-13, Rao, R. V. 1990. Natural decay resistance of neem wood. J. Indian Acad. Wood Sci. 21:19-21 Rodrigues, A. M.S. D. Stien, V. Eparvier, L. S. Espindola, J Beauchéne, N. Amusant, N. Leménager, C. Baudassé, and L. Ragu, 2012. The wood preservative potential of long-lasting Amazonian wood extracts. Ir. Bioderer. Blodegrad. 75:146-149, Ssjap, A.M. L.T. Lardizabal,F.B. Ahmad, and M. H, Sabri, 2006 Feeding response of subterancan temite, Coptotermes curvignathus soptera: Rhinotermitdae) to Azadirachta excelsa (Meliaceae) extractives and is timber, Sociobiology 48:487-455, Seri, M, ML Ishida, K. Nakata, M. Kim, and S. Takahashi, 1992, Antifeeding poteney of neem (Azediracta indica) extractives and limonoids against termite (Rericultermes speratus). J. Pestic. Se 17267-2773, ‘Standards Ausralia 2008, Specification for preservative treatment. Part 1: Sawn and round timber. AS. 1604.1-2005. Standards Australia, Sydney, Syoluna, A.A. Y. Banana, and G. Nakabonge. 2012. Efficiency of ‘natural wood extractives as wood preservatives aginst temite slack. Maderas Ciene. Tecnol. 142): 188-163, \Venmala D- and H.-C. Nagaveni, 2008, Evaluation of copperised ‘aahew nutshell liquid and neem oil as wood preservatives. IRGIWP! (05-30368. The Intemational Research Group on Wood Protection, Stockholm, Yashroy, R. C. and P. K. Gupta, 2008, Neem-seed oil ax termite repellent Toscol, dnt. 1227-28 MACHADO ET AL.